Detecting DENV in Urine and Saliva

C6/36 cells were used in attempt to isolate DENV from urine and saliva samples. Before inoculation, urine samples were dialyzed and concentrated in PBS using the 100K Microsep ultrafiltration system (Pall, USA) and then filtered through 0.2-μm membrane (Nalgene Thermo Scientific, USA). Saliva diluted in VTM as well as urine samples were diluted 1/2 with L15 Leibovitz Medium (Sigma Aldrich, Germany) containing 2% of fetal calf serum (Gibco Life Technology, USA). Final volumes of 300 µl of diluted specimens, or controls, were inoculated into 12-well plates containing 100% confluent C6/36 cells and incubated for 1 hour at 28°C. After incubation, 1.7 ml of medium was added to each well and the plates were incubated at 28°C. After 7 days, cells were harvested and DENV was detected by an immunofluorescence assay using serotype-specific monoclonal antibodies as described previously [33 (link)]. For samples that tested negative, two additional passages on C6/36 were performed before concluding that DENV did not replicate.
Our positive controls consisted of urine and saliva specimens obtained from healthy individuals as well as VTM spiked with infectious virus at a final concentration of 3 to 7 log10 cDNA copies/ml. Spiked urine controls were dialyzed and concentrated as for the patient’s urine samples.